Hydrogen sulfide (H2S) has been recently accepted as the third member of the gasotransmitter family that includes nitric oxide (NO) and carbon monoxide (CO). Due to homologous biological functions to NO, we posit that H2S has a critical role in estrogen-induced uterine vasodilatation. Endogenous H2S is mainly produced by metabolizing L-cysteine via two key enzymes: cystathionine ?-lyase (CSE) and cystathionine ?- synthase (CBS). We have obtained compelling data showing that estrogens stimulate CBS but not CSE expression, in ovine uterine artery (UA) endothelium and smooth muscle in vitro and in vivo as well as human UAs. Inhibition of uterine artery endothelial and smooth muscle cell H2S production by a CBS inhibitor attenuated estrogen-stimulated endothelial NO synthase (eNOS) activation in UA endothelial cells in vitro and the vasodilatory effects of estrogen on KCl-preconstricted UAs in rats. Thus, in this competitive renewal application we propose to test a novel hypothesis that endogenous H2S production by enhanced CBS expression in UA endothelium and smooth muscle functions as a novel vasodilator for mediating estrogen actions on uterine endothelium via interactions with endothelial eNOS-NO. This conjecture will be tested by four specific aims with comprehensive biochemical, cellular, molecular, physiological, and pharmacological approaches using in vitro cell culture models of UA endothelial and smooth muscle cells and ex vivo studies of various artery samples collected from sheep estrogen replacement therapy models and human UAs associated with different estrogens status from hysterectomy. Aim 1 will determine if estrogens stimulate H2S production via upregulating endothelial and smooth muscle CBS expression in UA but not systemic arteries from animal models ex vivo. Aim 2 will determine the mechanisms by which estrogens utilize to stimulate CBS expression and H2S production in UAEC vs. UASM cells in vitro. Aim 3 will determine if UASM and endothelium-derived H2S functions as a mediator for estrogen activation of the eNOS-NO pathway in endothelial cells. Aim 4 will determine if endogenous H2S mediates vascular reactivity to estrogens in human UAs from hysterectomies of post- and pre-menopausal (both secretory and proliferative phases) as well as late pregnant women. The proposed studies are novel because they are entirely different from the existing literature and they will determine for the first time whether a new vasodilator H2S mediates estrogen-induced uterine vasodilatation and the underlying mechanisms. Thus, data obtained will significantly improve our cellular and molecular understanding of estrogen and uterine blood flow biology; this, in turn, will drive the development of new strategies for combating preeclampsia, intrauterine uterine restriction, and other pregnancy failures. New data obtained will also shed lights on the understanding of the cardiovascular protective effects of estrogens.